1. Field of the Invention
The present invention is broadly concerned with polymer-metal ion composites and methods for reversibly binding target compounds using such composites. More particularly, the invention pertains to stable composites comprising an amorphous polymer matrix having a plurality of channels with metal ion-ligand complexes immobilized therein. The metal complexes include a metal ion, preferably an ion of a transition metal, having a ligand bonded to the metal ion and the polymer matrix. The metal complexes are dispersed within the polymer matrix so that the metal ions are preferably at least about 8 xc3x85 apart. The composites are formed by polymerizing a polymeric moiety with a metal-ion ligand complex template in the presence of a solvent. The template preferably further includes a spacer ligand which is selected based upon its size or properties and which can be removed after polymerization to yield a composite having a particular chemical and/or physical environment around the metal sites. In the methods of the invention, the composites are contacted with a target compound under conditions for binding the compound. The composites and methods are particularly useful for reversibly binding compounds selected from the group consisting of oxygen, carbon monoxide, carbon dioxide, compounds having an atom of P, S, or N, and mixtures thereof.
2. Description of the Prior Art
The function of metal ions in biomolecules is controlled by two interrelated structural features: the structure of the metal ion coordination sphere which includes the geometric relationship of metal-bound ligands; and the molecular architecture of the metal binding site that controls the secondary coordination sphere (or microenvironment) about the metal ion. While the role of the former is well known in directing the activity of metalloproteins, the importance of the latter cannot be overlooked. Microenvironments about the metal ion active sites which are induced by the protein structure regulate several properties, including hydrophobicity, polarity, electrostatics, solvation, and the dielectric constant. Furthermore, the morphology of the metal active site in metalloproteins can govern the accessibility of substrates by the metal ions. Protein-created microenvironments thus have a significant role in controlling the reactivity of the metal ions.
The effects of the microenvironment on the functions of metal ions in proteins is illustrated by the diverse activity of heme-containing proteins. (Dawson, J. H., Science, Vol. 240, p. 433, (1988); Ortiz de Montellano, P. R., Acc. Chem. Res., Vol. 20, p. 289, (1987)). In hemoglobin and myoglobin, the steric constraints and hydrogen bonding capacity of the distal side of the heme pocket has a significant effect on the oxygen binding properties of these proteins. (Suslick, K. S. et al., J. Chem. Educ., Vol. 62, p. 974, (1985)). In the oxygenases and peroxidases, the functions of enzymes are affected greatly by the various protein environments that house the catalytic iron heme moieties. For example, cytochrome P450 (a monooxygenase) and chloroperoxidase (which halogenates substrates) have identical heme active sites with axially bound thiolates, yet their functions are vastly different. (Dawson, J. H., Science, Vol. 240, p. 433, (1988)).
Protein structure also controls other properties necessary for metal ions to function in biomolecules. In most cases, the active sites are located within the interior of the proteins, isolated from each other to prevent undesirable interactions. For example, in human hemoglobin the four heme dioxygen binding sites are isolated from each other by the globin, and the closest distance between heme sites is 25 xc3x85. (Perutz, M. F. et al., Acc. Chem. Res., Vol 20, p. 309, (1987)). This is imperative for reversible O2 binding because if the heme sites were allowed physical contact, either by intra or intermolecular pathways, the four-electron auto-oxidation of O2 would occur, leading to thermodynamically stable xcexc-oxo bridge iron species. In hemoglobin, like many metal ion-containing proteins, access by external ligands to the metal sites is provided by channels that connect the active sites to the surface of the proteins. The channel structure, while providing a means of entry into the active sites, can also aid in orienting substrates as they approach the metal ion as well as assist in the selection of substrates.
In the past, there has been great interest in developing synthetic systems that mimic the structural, physical, and functional properties of the metal ion sites found in proteins. (Ibers, J. A. et al., Science, Vol. 209, p. 223, (1980); Karlin, K. D. Science, Vol. 261, p. 701, (1993)). One approach to examine the role of microenvironments in the functions of metal ions within proteins is to simulate various architectural features in low molecular weight systems. (Karlin, K. D., Science, Vol. 261, p. 701, (1993)). Design features found in proteins have been incorporated into organic ligand systems to help direct the chemistry at the metal centers in solution. The reversible binding of O2 to synthetic iron porphyrin is one example where the design of organic ligands can dictate the reaction chemistry at a distant metal site. (Suslick, K. S. et al., J Chem Educ., Vol. 62, p. 974, (1985); Momenteau, M. et al., Chem. Rev., Vol. 94, p. 659, (1994)). The picket-fence iron porphyrin of Collman was the first synthetic heme to reversibly bind O2 in solution at room temperature by preventing the intermolecular iron oxygen interactions that lead to xcexc-oxo bridge iron species. (Collman, J. P., Acc. Chem. Res., Vol. 10, p. 265, (1977)). A variety of other porphyrins and non-porphyrin ligands have since been designed containing cavity motifs that, when metallated with iron, are capable of forming Fe-O2 adducts. (Suslick, K. S. et al., J. Chem Educ., Vol. 62, p. 974, (1985); Momenteau, M. et al., Chem. Rev., Vol. 94, p. 659, (1994); Jones, R. D. et al., Chem. Rev., Vol. 79, p. 139, (1979); Busch, D. H. et al., Chem. Rev., Vol. 94, p. 585, (1994)). In addition, other notable examples where ligand design has aided in mimicking biological function in synthetic systems include: the specific recognition of metal ions; (Cram, D. J., Angew. Chem., Int. Ed. Engl., Vol. 27, p. 1009, (1988); Lehn, J. M., Angew. Chem., Int. Ed. Engl., Vol.27, p. 89, (1988); Pedersen, C. J., Angew. Chem., Int. Ed. Engl., Vol. 27, p. 1021, (1988)) the acceleration of the rates of chemical reactions; (Breslow, R., Science, Vol. 218, p. 532, (1982)) and in artificial receptors that show strong and selective binding of organic substrates (Hamilton, A. J., Chem. Educ. Vol. 67, p. 821, (1990); Diederich, F. J., Chem. Educ. Vol. 67, p. 813, (1990), Tjivikua, T. et al., J. Am. Chem. Soc. Vol. 112, p. 1250, (1990); Webb, T. H. et al., J. Am. Chem. Soc., Vol 113, p. 8554, (1991)). Finally, template copolymerization methods have been used extensively to generate systems that selectively bind analytes. Wulff, G., Angew. Chem., Int. Ed. Engl., Vol. 34, p. 1812 (1995); Mosbach, K., Trends Biotech., Vol. 19, p. 9 (1994); Shea, K. J., Trends Poly. Sci., Vol. 2, p. 166 (1994). In most cases, these molecular systems use a combination of morphological control of a binding cavity and weak bonding interactions to guide the recognition process.
Another approach simulating the site isolation properties of metalloproteins is to attach synthetic metal complexes onto the surface of solid supports. For example, embedding the diethyl ester of heme in a hydrophobic matrix of polystyrene and 1-(2-phenylethyl)-imidazole permits the Fe(II) sites of the heme to reversibly bind O2. (Wang, J. H., J. Am. Chem. Soc., Vol. 80, p. 3168, (1958); Wang, J. H., Acc. Chem. Res., Vol. 3, p. 90, (1970)). It has also been reported that crosslinked polystyrene containing attached imidazole ligands can coordinate Fe(II) tetraphenylporphyrin (FeIITPP). (Collman, J. P. et al., J. Am. Chem. Soc., Vol. 95, p. 2048, (1973)). However, this matrix is too flexible (or the sites are not sufficiently dispersed throughout the matrix) to prevent the formation of (FeIIITPP)2O. In a related system, FeIITPP has been attached to a rigid silica gel support that was modified with 3-imidazolylpropyl groups. (Leal, O. et al., J. Am. Chem. Soc., Vol. 97, p. 5125, (1975)). Reversible O2 binding to the Fe sites was observed, but that binding was weak. At xe2x88x92127xc2x0 C., the binding was irreversible and a P1/2(O2) of 230 torr was measured at 0xc2x0 C. The estimated P1/2(O2) for hemoglobin at 0xc2x0 C. is 0.14 torr.
Other types of matrices have been used to immobilize metal complexes for the purpose of reversible binding of O2, including the encapsulation of iron porphyrins in dendrimer cages (Aida, T. et al., Chem. Commun., Vol. 1, p. 1523, (1996); Collman, J. P. et al., Chem. Commun., Vol.2, p. 193, (1997)) and membranes, (Tsuchida, E. et al., J. Chem. Soc., Dalton Trans., p. 2465, (1993)) as well as the immobilization of cobalt Schiff base complexes in zeolite cages. (Howe, R. F. et al., J. Phy. Chem., Vol. 75, pg 1836, (1975); Herron, N., Inorg. Chem., pg. 4714, (1986); De Vos, D. E. et al., J. Am. Chem. Soc. Vol. 116, pg 4746, (1994). The dendrimer porphyrins have the ability to stabilize Fexe2x80x94O2 adducts. (Diederich, F. J., Chem. Educ. Vol. 67, p. 813, (1990); Aida, T. et al., Chem. Commun., Vol. 1, p. 1523, (1996); Collman, J. P. et al., Chem. Commun., Vol. 2, p. 193, (1997)). The zeolite systems use a xe2x80x9cship in the bottlexe2x80x9d protocol to assemble the Co(II) Schiff base complexes inside zeolite cages. The limitations of this technique are in the difficulty of matching the size and shape of the metal complex to that of the preformed cage as well as the inability of external gases to access the metallated sites within the interior of the zeolite. While, reversible binding has been observed in these systems, the quantity of Co sites involved in the binding is generally less than 25%. (Tsuchida, E. et al., J. Chem. Soc., Dalton Trans., p. 2465, (1993)). Thus, these prior art systems do not sufficiently isolate the metal sites from each other, and they generally utilize only a small percentage of the metal sites in the binding of the target compound.
The instant invention overcomes these problems by providing stable composites comprising immobilized metal complexes in porous polymeric hosts useful for reversibly binding target compounds. In more detail, the composites of the invention comprise an amorphous polymer matrix which includes a substitution-inert metal complex comprising a metal ion bonded to a ligand which in turn is bonded to the backbone of the matrix. Preferably, the composites have a plurality of spaced-apart metal complexes and a plurality of channels throughout the polymer matrix for allowing the target compounds to access the metal sites.
Advantageously, the metal ions are used to organize the desired functional groups within the polymer around the metal ion. The metal ion of the complex is preferably an ion of a transition metal, such as an ion of Co(III). The metal complexes should be spaced apart within the polymer matrix at such a distance that at least about 50%, preferably at least about 70%, and more preferably at least about 80% of the metal ions are at least about 8 xc3x85 from any other metal ion in the composite. In one embodiment, the metal ions are arranged within the polymer matrix so that at least about 50%, preferably at least about 70%, and more preferably at least about 80% of the metal ions are at least about 12 xc3x85 from any other metal ion. The channels within the polymer matrix preferably have an average diameter of from about 60-120 xc3x85, and more preferably from about 80-100 xc3x85.
While any polymeric moiety (monomer, dimer, trimer, oligomer, etc.) which produces rigid, crosslinked polymers when heated or exposed to light is suitable for forming the polymer matrix of the composites, the preferred moieties are organic and include a vinyl group. Particularly preferred polymeric moieties are those selected from the group consisting of divinyl benzene, dimethacrylates, diacrylamides, and mixtures thereof. If necessary, an initiator, such as azobisisobutyronitrile (AIBN), may also be used with the polymeric moiety in order to generate a free radical reaction.
The composites of the invention should include from about 5-20% by weight metal ion, and preferably from about 3-7% by weight, based upon the total weight of the composite taken as 100% by weight. Preferably, the composites include from about 80-97% by weight of the combination of polymer matrix and ligands, and preferably from about 90-97% by weight, based upon the total weight of the composite taken as 100% by weight.
The ligand moiety of the metal complex should be covalently bonded to the metal ion and covalently bonded to the polymer matrix. Preferably, the portion of the ligand bonded to the polymer matrix comprises a vinyl group, with a particularly preferred ligand being a styrene-modified salen ligand. During polymerization, the composites further include xe2x80x9cspacerxe2x80x9d ligands. After polymerization, these spacer ligands can remain in the composite or can be removed from the metal complex to yield the desired architecture around the metal site. Those skilled in the art will appreciate that these spacer ligands can be chosen to have particular properties or sizes so that the positioning of the polymer matrix and of the functional, non-spacer ligands within the polymer matrix is controlled. Furthermore, the metal ion used during polymerization can be removed and replaced with a different metal ion depending upon the desired binding properties of the polymer. The spacer ligand is covalently linked to the metal ion but not bonded to the polymer matrix. The stability constant (as determined spectrophotometrically at room temperature) of the spacer ligand bonded to the metal ion is from about 102-1030 Mxe2x88x921, and preferably from about 107-1020 Mxe2x88x921. A preferred spacer ligand is a dimethylaminopyridine ligand.
The methods of forming the composites of the invention are schematically depicted in FIG. 1. A substitution inert metal complex template having the desired ligands attached thereto (including both the functional, non-spacer ligands as well as the spacer ligands, the latter of which will generally be removed to provide the desired architecture around the metal ion) is attached to the metal ion. The template is then polymerized with a polymeric moiety (such as divinyl benzene, dimethacrylates, or diacrylamides) in the presence of a porogenic agent (solvent) capable of dissolving the moiety monomers before polymerization, thus forming the plurality of channels within the polymer matrix. Some preferred solvents include methanol and dimethylforamide (DMF). The spacer ligands can then be removed by exposing the composites to a suitable chelating agent (such as cyanide or EDTA) which will break the bonds between the metal ion and the functional groups which are bonded to the polymer matrix, thus removing the metal ion and the spacer ligands. The same metal ion or a different metal ion can then be returned to the site without the spacer ligands. One suitable way of returning the metal ion is by way of a metal salt along with a solvent for that salt. The resulting composite is then preferably formed into a particle size of from about 75-125 xcexcm, depending upon the particular application. FIG. 2 schematically depicts the final formed composite with the metal ions 10 dispersed within the polymer matrix 12 and bound to the matrix by ligands 14.
In accordance with the methods of the invention, the composites can be used to bind target compounds by contacting the composites with the target compound under conditions for binding that compound. Preferably, the contacting step is carried out under ambient conditions. The target compound can then be removed from the composite by heating the compound-containing composite, preferably to a temperature of about 120xc2x0 C. The contacting step results in at least about 50%, and preferably at least about 80%, of the metal ions of the composite having a target compound bound thereto. When stored in an ambient environment, at least about 50%, and preferably at least about 80%, of the metal ions having a target compound bound thereto during said contacting step will still have a target compound bound thereto after about 7 days.
Target compounds which can be bound by the composites include those selected from the group consisting of oxygen, carbon monoxide, carbon dioxide, compounds having an atom of P, S, or N, and mixtures thereof The instant invention is particularly useful for reversibly binding NO and oxygen. Thus, the composites of the invention can be used to remove NO generated from the combustion of fossil fuels. Furthermore, upon the binding of NO with the composite, the color of the material changes from orange to green, making the composites suitable for use in NO sensor technology. The development of NO sensors is important in probing the biological functions of NO which is both a vasodilatory messenger and an endothelial-derived relaxing factor and plays a key role in cellular communication. With modification of the polymeric surface of the composites, they can be attached to an optical fiber for in vivo NO detection in the presence of other gaseous analytes.